Re: [ccp4bb] Problems in purification

[Roger Rowlett]

I generally find it is possible to purify most overexpressed proteins, even those at low levels of _expression_, with a combination of IEX and HIC methods. We use step-gradients to do our routine purifications, but may use gradients for polishing. If running gradients to polish partially pure samples, take care to run fairly long ones to achieve decent resolution. We routinely polish protein preps on Q-sepharose (Mono-Q should be even better) with at least 10 CV gradients over a narrower range of NaCl concentrations, maybe 0-0.5 M or even smaller. We favor butylsepharose for HIC, as it is less sticky and more selective in our hands. Again, a long gradient (10 CV or more) over a narrower range of salt concentrations will be most effective. We always use GEC for a final polish and desalting. Cheers. On 8/26/2010 8:24 AM, ganesh pathare wrote: Dear all,   I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein _expression_ levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it contains lot of impurities. I varied the salt concentrations out of which I could get optimal results at 20 mM NaCl concentration but still the amount of impurities was more. After affinity purifications I used Ion exchange chromatography using MonoQ column (25 mM tris pH 7.5,  NaCl 0 to 1M) which could not seperate the protein from the impurities. I also tried using Hydrophobic interaction chromatography (Resource Ether, Phenyl sepharose, Resource Isopropyl) instead of ionexchange chromatography, which resulted in better purification of the protein, but the problem is I get very less protein after this step and there are still two major impurities. The buffer conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH 7).     I would be very greatful if someone could help me in this concern. Thanks in advance.   Regards, Ganesh -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu